Krutkina Ekaterina, Klaiman Daniel, Margalit Tamar, Jerabeck-Willemsen Moran, Kaufmann Gabriel
Department of Biochemistry, Tel Aviv University, Ramat Aviv, 69978, Israel.
NanoTemper, Flössergasse 4, 81369 München, Germany.
Virology. 2016 Jan;487:260-72. doi: 10.1016/j.virol.2015.10.012. Epub 2015 Nov 10.
The anticodon nuclease (ACNase) PrrC is silenced by a DNA restriction-modification (RM) protein and activated by a phage T4-encoded restriction inhibitor. The activation is driven by GTP hydrolysis while dTTP, which accumulates during the infection, stabilizes the active form. We show here, first, that the ABC-ATPase N-domains of PrrC can accommodate the two nucleotides simultaneously. Second, mutating a sequence motif that distinguishes the N-domain of PrrC from typical ABC-ATPases implicates three residues in the specificity for dTTP. Third, failure to bind dTTP or its deprivation hastened the centrifugal sedimentation of PrrC, possibly due to exposed sticky PrrC surfaces. Fourth, dTTP inhibited the GTPase activity of PrrC, probably by preventing GDP from leaving. These observations, correlated with relevant traits of a related ACNase, further suggest that PrrC utilizes GTP at canonical ABC-ATPase sites and binds dTTP to distinct sites exposed upon disruption of the ACNase-silencing interaction with the RM partner.
反密码子核酸酶（ACNase）PrrC被一种DNA限制修饰（RM）蛋白沉默，并被噬菌体T4编码的限制抑制剂激活。这种激活由GTP水解驱动，而在感染过程中积累的dTTP会稳定活性形式。我们在此表明，首先，PrrC的ABC-ATPase N结构域可以同时容纳这两种核苷酸。其次，突变一个将PrrC的N结构域与典型ABC-ATPases区分开来的序列基序，发现三个残基对dTTP具有特异性。第三，未能结合dTTP或其缺失加速了PrrC的离心沉降，这可能是由于PrrC表面暴露有黏性。第四，dTTP可能通过阻止GDP离开而抑制了PrrC的GTPase活性。这些观察结果与相关ACNase的相关特性相关，进一步表明PrrC在典型的ABC-ATPase位点利用GTP，并将dTTP结合到与RM伙伴的ACNase沉默相互作用被破坏时暴露的不同位点。
